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Diagnostic Technologies and Molecular and Cellular Profiling: Biomarker Assay Development, Validation, and Qualification

Predicting temozolomide resistance with MGMT RNA expression in metastatic melanoma

Duke University Medical Center, Durham, NC

Abstract

A2

Introduction: Temozolomide (TMZ) is an effective agent in the treatment of metastatic melanoma whose in-vivo anti-tumor activity correlates directly with O6-methylguanine-DNA methyltransferase (MGMT) activity. In this study, we examined whether MGMT RNA expression could serve as a surrogate marker for MGMT activity. The use of a PCR-based assay could facilitate clinical trials involving TMZ, particularly when only small tumor samples are available.Methods: The in-vitro sensitivities of 40 melanoma cell lines to TMZ was determined using a high-throughput, cytotoxic assay (Promega CellTiter Blue assay). Resistance to TMZ was defined as 55% sensitivity index across 0-3mM. MGMT RNA expression and activity were measured using quantitative PCR and HLPC, respectively. High MGMT RNA level was defined as 0.15 relative to MGMT expression in HT29 cell line. High MGMT activity was defined as 150 fmol/mg. Correlation analyses were performed where Pearson's correlation coefficients were determined for MGMT expression and activity against TMZ sensitivities. Qualitative analyses were also performed using the criteria outlined above. Mismatch repair status of the cell lines were determined by testing for microsatellite instability using PCR and ABI Genescan 3100 analyzer (Applied Biosystem) with five mononucleotide markers (BAT-25, BAT-26, NR-21, NR-22, and NR-25).Results: There was a strong correlation between MGMT RNA expression and MGMT activity (correlation coefficient 0.84, p-value <0.0001). Using our criteria, overall accuracy of predicting TMZ sensitivity based on MGMT RNA expression was the same when compared to using MGMT activity (70%). Furthermore, the overall accuracy increased to 79% and 74%, for RNA expression and activity, respectively, when cell lines without MGMT activity/level were excluded. Only one cell line (PR Mel) was found to be mismatch repair deficient, which was resistant to TMZ as expected.Conclusion: The accuracy of predicting TMZ sensitivity with MGMT RNA expression is comparable to using MGMT activity. In a subset of cell lines in which MGMT was not predictive of TMZ sensitivity suggest other resistance mechanism at work, especially in cell lines with minimal MGMT activity/level. Furthermore, mismatch repair deficiency did not contribute significantly to the non-MGMT resistance mechanism in our cell lines.